1、Quantitative HPLC Analytical Method forTetramethylpyrazine Derivative TBN Student NameMAK SAU HANGStudent No.MajorSupervisorProfessor Wang, YuqiangDate(dd/mm/yyyy)暨南大學(xué)本科生畢業(yè)論文論文題目川芎嗪衍生物TBN的HPLC含量測(cè)定方法學(xué)院國(guó)際學(xué)院學(xué)系藥學(xué)系專(zhuān)業(yè)藥學(xué)姓名學(xué)號(hào)指導(dǎo)教師Statement of OriginalityI hereby declare that the thesis presented is the resul

2、t of research performed by me personally, under guidance from my supervisor. This thesis does not contain any content (other than those cited with references) that has been previously published or written by others, nor does it contain any material previously presented to other educational instituti

3、ons for degree or certificate purpose to the best of my knowledge. I promise that all facts presented in this thesis are true and creditable. Signed:_ Date：Quantitative HPLC analytical method for tetramethylpyrazine derivative TBNAbstract:A high-performance liquid chromatography (HPLC) method for a

4、new tetramethylpyrazine derivative, TBN, was developed. Analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit ofquantification (LOQ) were determined under the guideline of International Conference on Harmonization Q2B1. TBN was anal

5、yzed by RP-HPLC with C18 column using methanol-water (35:65) as mobile phase. The flow rate is 0.8 ml/min and the detector was set to 295 nm. The linearity of calibration curve is good (r2 > 0.999) and the LOD and LOQ were 12.096ng/ml and 40.32ng/ml respectively. The relativestandard deviation (R

6、SD) of precision and accuracy were 0.1245 % and 0.6895%, respectively, and thesample recovery was 99.08 % (RSD: 0.40 %). This method is reliable and easy for TBN analysis.Key Words:tetramethylpyrazine derivative, TBN, HPLC川芎嗪衍生物TBN的HPLC含量測(cè)定方法摘要：目的：建立川芎嗪衍生物 (TBN) 高效液相含量測(cè)定方法。方法：選用 C-18 色譜柱；甲醇-水 (35:65

7、)；流速：0.8 ml/min；檢測(cè)波長(zhǎng)為295 nm。結(jié)果：TBN 在12.42- 310.5 mg/ml 范圍內(nèi)線性關(guān)系良好 (r2 > 0.999) ；定量限和檢測(cè)限分別為 12.096 ng/ml 及 40.32 ng/ml；精密度及準(zhǔn)確度 RSD 分別為 0.1245 % 及 0.6895%；加樣回收率結(jié)果為 99.08 % (RSD: 0.40 %)。結(jié)論：本方法簡(jiǎn)便可靠，可作為T(mén)BN之定量分析方法。關(guān)鍵詞：川芎嗪衍生物，TBN，高效液相Contents1. Introduction32. Materials32.1. Chemicals32.2. Apparatus33.

8、Experimental Method33.1. TBN standard preparation33.1.1. Column chromatography for TBN purification33.1.2. Semi-preparative column chromatography33.2. Development of HPLC method for TBN33.2.1. lmax determination33.2.2. Optimization of HPLC conditions for TBN33.3. Validation of HPLC method33.3.1. Spe

9、cificity33.3.2. Linearity33.3.3. Precision33.3.4. Limit of Quantification and Limit of Detection33.3.5. Range33.3.6. Accuracy33.3.7. Stability33.3.8. Recovery33.3.9. Content Assay34. Results and Discussion35. Conclusion3Acknowledgement3References31. IntroductionTBN is a novel compound developed by t

10、he Institute of New Drug Research at the Pharmacy College, Jinan University.TBN has been shown to be antioxidative and thrombolytic, and is under development as a treatment for ischemic stroke 2. TBN is a derivative of 2,3,5,6-tetramethylpyrazine (TMP), which is the main active ingredient of Ligusti

11、cumwallichii Franchat (Chuan Xiong), formed by conjugating TMP and a nitrone moiety (see Figure 1) . TMP is used to treat ischemic stroke in China for many years which was found beneficial in inhibiting platelet aggregation 3, lysing blood clots4, blocking calcium entry 5 and scavengingreactive oxyg

12、en species (ROS)6. While retaining the thrombolytic activity of TMP, the nitrone added to TMP (i.e. TBN) had been proved to provide a strong antioxidative activity.Nitrones are useful as therapeutic agents for neural and systemic diseases such as atherosclerosis, septicemia, stroke, and Alzheimers d

13、isease 7. (1) (2) (3)Figure 1 Structure ofTMP (1); Nitrone (2); TBN (3)As a potential new drug, series of researches like pharmacology, toxicology, pharmacokinetics, pharmacodynamics are needed.Therefore, quality analysis and control on TBN is necesary. First of all, a quantitative method of TBN had

14、 to be developed. HPLC method for analyzing chemical compound is quick, simple and reliable. Therefore we chose it as the analyzing method for TBN. A reliable HPLC method should be able to separate the sample from its impurities completely and can be validated properly, so called the Methodology.The

15、 validation of developed HPLC method1, 8 included several parts which are linearity, precision, range, limit of quantification and limit of detection, accuracy, stability, specificity, and recovery. The content assay would be done after the validation of method. However, reference standard of the su

16、bstance to be tested is needed in some items of the HPLC method validation.For this case, TBN is a new compound and its reference standard is unavailable. Therefore, in this paper, a relatively pure sample was used as a reference standard. Hence, to obtain a relatively pure TBN sample, a purificatio

17、n of TBN was also done in this research.The target of this research is to establish an easy and reliable HPLC method for TBN analysis, used in routine quality control of its related studies. After the establishment of TBN reference standard, this HPLC method can be applied directly to quantify TBN c

18、ontent. In this research, there were three main parts: TBN standard preparation, HPLC method establishment, and Method validation.2. Materials2.1. Chemicalsl TBN, prepared by the Institute of New Drug Research at Pharmacy College, Jinan University; l Petroleum ether, ethyl acetate, acetone and dichl

19、oromethane (Analytical reagents) purchased from FUYU Refined Chemical Products Ltd. ;l Reagent graded silica gel (200-300 mesh) purchased from Branch of Qingdao Haiyang Chemical Plant;l Methanol (HPLC grade) purchased from Jiangsu Hanbon Sci. & Tech. Co., Ltd. ;l Double-distilled water provided

20、by the Institute;l Sodium dihydrogenphosphate and disodium hydrogenphosphate (Analytical reagents), purchased from Guangzhou Chemical Reagent Factory. 2.2. Apparatusl Glass Chromatographic column;l Thin layer chromatographic silica gel plates;l Rotary Evaporator (EYELA N-1001) and Digital Water Bath

21、(EYELA SB1000) ;l Vacuum drying oven(DZF-6050);l Ultrasonic Cleaners(KQ-250E);l Melting Point Measuring Instrument(SGW X-4);l Shimadzu UV-VIS Spectrophotometers (UV-2450)l Shimadzu-10AT HPLC and SPD-10AVP UV-VIS Detector;l LUBEX Kromasil C18 column (5 m 100Å 250 mm´4.6 mm);l semi preparati

22、ve HPLC column (VYDAC RP C18 90A PHARMACEUTICAL);l Electronic balance(ACCULAB ALC110.4);l Other apparatus: volumetric flasks, conical flasks, pipette, micro filters and membranes (0.45 mm). 3. Experimental Method3.1. TBN standard preparationTBN is a white crystal which is water soluble, with molecul

23、ar weight of 221 g/mol. Due to the difference in purity between each batches of production, the physical properties of TBN samples are varied from one another. In this part, relatively pure TBN sample would be prepared and then used in the validation of HPLC method as reference standard.3.1.1. Colum

24、n chromatography for TBN purification(1) TLC methodUse ethyl acetate as the solvent to dissolve TBN sample, separate the sample components with different mobile phases.A. The continuous separating condition: petroleum ether-ethyl acetate (1:1)B. Modified condition 1: petroleum ether-ethyl acetate (4

25、:1)C. Modified condition 2: petroleum ether-acetone (5:1)Figure 2 TLC of TBN with different mobile phase (from left to right : A, B, C)(2) Column chromatographyFrom the results of TLC, method B and C were implemented in column. About 300 mg of TBN were used in each method. For method B, yellow semi

26、solid was obtained. Very pale yellow solid was obtained in method C. Hence, method C was chosen.Batch 2008.10.11 TBN (4.26 g) was put in column chromatography with the mobile phase petroleum ether-acetone (5:1) and 2.88 g of purified TBN was yielded. Melting point was tested 77.3.1.2. Semi-preparati

27、ve column chromatographySince there were trace amount of impurities in the purified TBN from 3.1.1, semi-preparative column chromatography was performed for further purification.One hundred mg of TBN was used this time, and a methanol-water (35:65) system was used as a mobile phase. However, Shimadz

28、u-10AT HPLC is not designed for semi-preparative column, too much solvent was used and too few TBN have been purified. This method is not feasible for producing sufficient purified TBN. And for the TBN purified, since there was too much water in the mobile phase that was difficult to dry, only trace

29、 amount of TBN was obtained which was not enough for experimental use. With the result, the TBN purified from 3.1.1 was used as reference standard.3.2. Development of HPLC method for TBN3.2.1. lmax determinationBefore the procedure of HPLC, the lmax of TBN should be determined. With the UV-VIS Spect

30、rophotometer, the TBN sample solution was scanned in 200-400 nm for the determination of lmax.Method:TBN (0.01 g) from 3.1.1 was weighted and was dissolved in 10 ml volumetric flask with methanol. Dilute the solution to 50 mg/ml by using pipette and volumetric flasks.Scan the sample solution with th

31、e UV-VIS Spectrophotometer and record the result.Figure 3 UV spectrogram for TBNFrom the above result, 295 nm was chose for TBN detection.3.2.2. Optimization of HPLC conditions for TBNLUBEX Kromasil C18 column (5 m 100Å 250 mm´4.6 mm) was used in this research which is suitable for most or

32、ganic compound.Different ratio of mobile phase, flow rate, pH, have been used to determine the most suitable HPLC method for TBN. With the aim of separating the TBN from its impurities, TBN sample without purification (Batch 20081019) were used. The mobile phase were prepared daily and degassed by t

33、he ultrasonic cleanser for not less than 20 minutes.Table 1 Different HPLC conditions tested for TBNMethanolWaterDetector lFlow ratepH7030295 nm0.9-1.0 ml/minNo added60400.8-0.8 ml/minNo added50500.8-1.0 ml/min45550.8-1.0 ml/minNo added, 7.240600.8-1.0 ml/minNo added35650.8 ml/min3.3. Validation of

34、HPLC methodThe reference standard used in this part was purified in 3.1, which was assumed 98.5 % pure. The HPLC chromatogram of TBN reference standard showed about 99 % purity of TBN, but since the UV detector in HPLC cannot detector every component in the sample, therefore the percentage content o

35、f TBN is still unknown.3.3.1. SpecificityTwo methods have been used to evaluate the specificity of the established HPLC method: 1) Raw material; 2) Degraded sample 1.Method 1Weight precisely about 10 mg of TMP raw material, diluted in 10 ml volumetric flask, takes 2 ml into a 5 ml volumetric flask a

36、nd diluted to the mark, take 1 ml of the solution into a 10 ml volumetric flask, add 0.5 ml of the TBN standard solution and diluted to mark. The dilutions were done with mobile phase. Inject and record the chromatogram.Method 2Use the TBN standard solution stayed for 48 hours. Inject and record the

37、 chromatogram.3.3.2. LinearityThe method used in linearity here had been used in lots of literature 9-17.Weight 0.0630 g of TBN in 50 ml volumetric flask and dilute to mark. Purity of TBN was assumed 98.5 %, thus there were 0.0621 g of TBN in the standard. Concentration was equal to 1.242 mg/ml, thu

38、s 1242 mg/ml. (The standard solution of TBN)Measure precisely 0.1 ml, 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml individually into five 10 ml volumetric flasks and dilute to marks. All of dilutions were done by mobile phase. Inject the solution and record he peak areas. Plot a standard curve and calculate t

39、he r2.3.3.3. PrecisionMeasure 2.2 ml from the TBN standard solution (1242 mg/ml) into a 10 ml volumetric flask, diluted to the mark with mobile phase.Inject the TBN solution for 6 times continually and record the peak areas for evaluating the precision and repeatability1, 8-18.3.3.4. Limit of Quanti

40、fication and Limit of DetectionDilute the TBN standard solution (1242 mg/ml) gradually until the peak height equal to 10:1 and 3:1 to the baseline noise respectively1. 3.3.5. RangeThe standard curve obtained from linearity would give the range1, 8-18.3.3.6. AccuracySix portions of 1.5 ml TBN standar

41、d solution (1242mg/ml) were measured and diluted in six 10 ml volumetric flasks, named 1-6. Inject the solutions and record the peak areas15-18.3.3.7. Stability1.5 ml TBN standard solution (1242 mg/ml) was measured and diluted in 10 ml volumetric flask. The solution was injected at 0, 2, 4, 6, 8 hou

42、rs respectively after preparation, record the peak areas15-18. 3.3.8. Recovery(1) Content assay (for sample recovery1, 9-17)Use the batch 20081107 as sample.About 10 mg TBN was precisely weighted and dissolved in 10 ml volumetric flask with mobile phase solution to the mark. Measure 1 ml from the sa

43、mple solution and diluted in 10 ml volumetric flask with mobile phase. Inject the solution for 3 times and record the peak area. Apply to the standard curve and calculate the percentage content of the TBN sample.(2) Recovery testTBN batch 20081107 was used in this part as sample.MethodMeasure 3 ml f

44、rom the TBN sample solution (concentration: 109 mg/ml, 86.15% TBN content) to nine 10 ml volumetric flasks respectively.Measure the standard solution (concentration: 1242mg/ml) which was equivalent to 80 %, 100 % and 120 % of the TBN content in the sample solution respectively, and transfer to the v

45、olumetric flasks with TBN sample solution, 3 portions for each were prepared.Dilute the solution to the marks with mobile phase. Inject and record the peak areas.3.3.9. Content AssayAbout 10 mg TBN was weighted precisely and dissolved in 10 ml volumetric flask with mobile phase solution to the mark.

46、Repeat step 1 for total 4 batches of TBN.Measure 1 ml TBN solution from each of flasks and dilute in another 4 volumetric flasks with mobile phase solution to the marks.Inject the 4 batches of diluted solution to HPLC for 3 times each and record the peak area.Apply the result to the standard curve a

47、nd calculate the TBN content in each batch15-18.4. Results and DiscussionOptimization of HPLC conditions for TBNFigure 4 Methanol-water (35:65) flow rate: 0.8 ml/min (solution stayed for 48 hours)This HPLC method was performed in two Shimadzu-10AT HPLC machines of Pharmacy College and had similar re

48、sults. I decided to start the methodology experiment with this condition to see if the result would be valid.The HPLC method developed in this article was evaluated that is suitable for TBN analysis. Since the presence of impurity with similar TBN polarity, not only the ratio of mobile phase, but th

49、e flow rate has to be adjusted, hence the longer analyzing time in the method.From the chromatograms have been done, variation in the retention time of TBN was presence due to the small difference in the volume ratio of daily prepared mobile phase and the variation is between 24-27 min. Validation o

50、f HPLC methodSpecificityMethod 1Figure 5 Chromatogram of TBN+ TMPMethod 2Figure 6 Chromatogram of TBN standard solution stayed for 48 hoursMethod 2 was more significant here, as long as the study, there were no observable TMP in the TBN, but the HPLC method still proved feasible when TMP is present.

51、 From the results above, the HPLC method established was well separated with the impurities in TBN sample.LinearityTable 2 Results of linearityConcentration mg/mlPeak Area uV*s12.4287310462.104298987186.3013507016273.2420022885310.5023350922Figure 7 The standard curve was reliable, r20.999Besides th

52、e above result, there are two invalid results have been done during the research:Method 1.This method was used because it was used in an article published from Institute of Drug control18.About 10 mg TBN was weighted from TBN standard precisely, dilutes in 50 ml volumetric flask to the mark.Inject 2

53、, 4, 6, 8, 10, 12, 14, 16 ml precisely into HPLC, record the peak areas.Table 3 Results of method 1Injection Volume mlPeak Area uV*s217131154407336466280787887568301010976877121315832914151845861617755992Figure 8 Standard curve of method 1The result of this method was good in correlation coefficient

54、. However, the range of this curve was too narrow. Also, it is not accurate to use the microinjector for sampling. A sampler should be used.Method 2.This method was used commonly in the establishment of standard curve which have been used in most of articles I found9-17. The advantages of this metho

55、d are accurate, wide range in the curve, but it is somewhat more difficult to operate because the error comes from the solutions preparation would affect the correlation coefficient of the standard curve directly.0.0630 g of TBN was weighted in 50 ml volumetric flask and diluted to mark.Assumed puri

56、ty of TBN was 98.5 %, thus there was 0.0621 g of TBN in the standard. Concentration was 1242mg/ml.Measure precisely 0.1 ml, 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml individually into five 10 ml volumetric flasks and dilute to marks. All of dilutions were done by mobile phase.Table 4 Results of method 2 (1

57、st time)Concentration mg/mlPeak Area uV*s12.411106156262.0554848641124.119518987186.16513366944248.2217509484,Figure 11 The standard curve of method 2 which was unreliable because r2 0.999In the evaluation of linearity, 2 methods have been used, and only method 2 is valid. The invalid of method 1 were due to 1) the range of evaluation was too narrow (i.e. 8 folds); 2) the